Kurt Ahrén

Analysis of refractoriness to the effects of growth hormone on amino acid transport and protein synthesis in diaphragms of young normal rats.

The in vitro responsiveness of diaphragms from 18-day-old fasted rats to bovine GH (bGH) was studied after the administration of bGH in vivo by determining the accumulation of α-amino-isobutyric acid to measure amino acid transport and the incorporation of phenylalanine into diaphragm protein to assess protein synthesis. bGH in vivo induced a transient stimulation of both processes. Three hours after the in vivo administration of 50 μg bGH iv, the rates of protein synthesis and amino acid transport had returned to prestimulatory levels. Moreover, bGH in vitro (5 μg/ml) had no effect on the two processes at this time, i.e. the muscle was refractory to GH. Five and 7 h after the administration of 50 or 100 fig bGH in vivo, the α-amino-isobutyric acid transport system was completely refractory to bGH in vitro, in contrast to protein synthesis which showed a full response to bGH in vitro. This finding indicates that the duration of refractoriness of amino acid transport to GH was considerably longer than that...

Prostaglandin levels in preovulatory follicles from rabbit ovaries perfused in vitro.

Prostaglandin (PG) levels in follicular fluid from preovulatory follicles of rabbit ovaries perfused in vitro were measured in order to compare PG changes in this model system with those that occur in vivo and in isolated, LH-treated follicles in vitro. One ovary from each rabbit was perfused without further treatment (control). The other ovary was exposed to LH (0.1 or 1 microgram/ml) beginning 1 hour (h) after initiation of perfusion. Samples of perfusion medium were taken at frequent intervals for measurement of PGE, PGF, progesterone and estradiol 17 beta. The perfusions were terminated when the first ovulation occurred or appeared imminent as judged by changes in the size and shape of the follicles. Follicular fluid was then rapidly aspirated from all large follicles on both ovaries for PGE and PGF measurement. Ovulations occurred only in the LH-treated ovaries. Progesterone and estradiol levels were significantly elevated in the perfusion medium within 1 h of LH treatment in comparison to controls. PG levels in perfusion medium from the control and LH-treated ovaries were not different throughout perfusion and increased in both groups. In contrast, PG levels measured in follicular fluid from LH-treated ovaries were 4- to 5-fold greater than in fluid from control ovaries. It is concluded that ovulation induced by LH in this experimental model is accompanied by an increase in follicular PG levels similar to that seen in other in vivo and in vitro models. This difference in follicular PG levels between the LH-treated and control ovaries is, however, not reflected in the perfusion medium.

CONTROL OF STEROIDOGENESIS IN THE PREOVULATORY RAT FOLLICLE

Publisher Summary This chapter describes the control of steroidogenesis in the preovulatory rat follicle. Whole preovulatory follicles extirpated before the endogenous gonadotrophin surge—morning follicles—secreted predominantly estradiol and androstenedione, and very low amounts of progesterone. Addition of LH in vitro increased the secretion of all three steroids. Evening follicles, which are removed after the endogenous gonadotrophin surge, formed small amounts of androstenedione and estradiol when incubated in hormone-free medium, while progesterone secretion was markedly enhanced compared to the situation before the gonadotrophin surge. Addition of LH to evening follicles had no effect on androstenedione and estradiol secretion, while the hormone was able to enhance further the formation of progesterone. It is found that steroid secretion by follicles removed in the evening from rats where the endogenous gonadotrophin surge was blocked by Nembutal was similar to that of morning follicles both in absence and presence of LH in vitro . Addition of testosterone to isolated evening follicles markedly enhanced estradiol secretion, while addition of dihydrotestosterone or 17-OH-progesterone had no effect.

Effect of androgens on the biosynthesis of estradiol-17β by isolated periovulatory rat follicles

Abstract To analyse the mechanism for the earlier reported decline in estrogen synthesis by the periovulatory rat follicle, prepubertal rats injected with 8 IU PMS on day 28 were killed following the endogenous gonadotrophin surge (15.00–18.00) on day 30. Isolated preovulatory follicles were incubated for 2 h in a chemically defined medium. Steroids were measured by specific RIA methods. Follicles exposed in vivo to the gonadotrophin surge and extirpated 19.00–22.00 h on day 30 secreted significantly lower amounts of androstenedione, testosterone and estradiol-17β but significantly higher amounts of progesterone than did follicles extirpated from rats in which the gonadotrophin surge had been prevented by a Nembutal injection. Secretion of estradiol-17β by follicles isolated following the endogenous gonadotrophin surge remained low when LH, FSH or dibutyryl cyclic 3′,5′-AMP was added to the medium. However, the addition of testosterone (0.1–1 μg/ml) or androstenedione (1 μg/ml) to the incubation medium restored estradiol biosynthesis to values similar to those seen prior to gonadotrophin exposure. There was no effect of 5α-dihydrotestosterone or 17α-hydroxyprogesterone on the estradiol-17β synthesis. The results indicate that cleavage of the 17:20 sidechain rather than the aromatase enzyme limits estradiol synthesis in the periovulatory follicle following the gonadotrophin surge. It is suggested that the combined action of LH and FSH of the gonadotrophin surge might explain the lack of inhibitory effect on the aromatase enzyme recently reported by Katz and Armstrong (1976) 6–8 h after the injection of LH.

Comparison between the effect of luteinizing hormone and prostaglandin E1 on ovarian cyclic AMP

Abstract Isolated whole ovaries from 23–24 day-old rats were studied in order to compare the effects of prostaglandin E 1 (PGE 1 ) and luteinizing hormone (LH) on ovarian cyclic adenosine 3′,5′-monophosphate (cAMP) production. Both substances produced a dose-dependent accumulation of cAMP in the ovarian tissue as well as in the incubation medium. The release of cAMP to the incubation medium was considerable after long periods of incubation (60–120 min). Time-relationships for LH- and PGE 1 -effects were different. Maximal cAMP content in the tissue after addition of PGE 1 was seen already after 5–15 min of incubation whereas LH gave a maximal response after around 60 min. Accumulation of cAMP in the medium was approximately linear with time for both LH and PGE 1 . Addition of theophylline potentiated the action of PGE 1 and LH but did not change the time-courses of the effects. It is concluded that the accumulation of cAMP in the medium should be considered in studies with various in vitro types of ovarian preparations. It is also pointed out that the different time-courses of the LH- and PGE 1 -effects make the interpretation of additivity experiments difficult.

Control Mechanisms for the Synthesis of Glycogen in Striated Muscle

The key enzyme in the regulation of glycogen synthesis in muscle is the glycogen synthetase (UDP- glucose: α-1, 4-glucan α-4-glucosyltransferase; E C 2. 4. 1. 11). It is well-established that many hormonal and non-hormonal factors which under physiological conditions stimulate or inhibit the rate of glycogen synthesis produce their effects by influencing, directly or indirectly, the activity of this enzyme. It is also well-known that the glycogen synthetase enzyme from muscle tissue, as well as from all other mammalian tissues heretofore investigated, exists in two forms, one (D-form) dependent upon and another (I-form) independent of glucose-6-phosphate (G-6-P) for its activity. These two forms are interconverted by a mechanism involving phosphorylation of the I-form in presence of a synthetase I kinase, and dephosphorylation of the D-form by a synthetase D phosphatase. The per cent synthetase I, observed in a tissue at any one time represents a balance between the activity of the phosphatase, catalysing D→I conversion, and the kinase, catalyzing I→D conversion. If this balance is disturbed by, for instance, a partial or total inactivation of the kinase, then the opposing action of the phosphatase becomes dominant and there is a net increase in the I-form. On the other hand, if the phosphatase is inhibited, then the action of the kinase becomes dominant with an increase in the D-form as a result.

Sphingosine and psychosine, suggested inhibitors of protein kinase C, inhibit LH effects in rat luteal cells.

The possible involvement of protein kinase C on luteinizing hormone (LH) effects in dispersed rat luteal cells was investigated using two substances that have been reported to be protein kinase C inhibitors, sphingosine and psychosine. Sphingosine efficiently inhibited protein kinase C activity both in brain and luteal cytosol fractions. Both substances inhibited LH-stimulated cyclic adenosine monophosphate (cAMP) accumulation in a dose-dependent fashion with an LD50 at 3-7 microM (sphingosine) and 40 microM (psychosine). LH-stimulated progesterone production was also inhibited with an ID50 at 6-10 microM (sphingosine) and 40-100 microM (psychosine). The inhibition was not due to an increased phosphodiesterase activity since IBMX (3-isobutyl-1-methylxanthine, 0.1 mM) and RO 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, 0.1 mM) did not abolish the inhibitory effect of sphingosine. To study the mode of action of sphingosine, forskolin and cAMP analogues were tested. The effect of these substances on steroidogenesis was inhibited, as well as the forskolin-induced cAMP accumulation, by sphingosine. This study demonstrates a clear inhibition of LH-stimulated effects by sphingosine and psychosine. LH action in rat luteal cells is discussed in relation to protein kinase C and the possible mode of sphingosine action.

Prostaglandin F2a inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F2 alpha (PGF2 alpha). The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF2 alpha. A dose-dependent inhibition by PGF2 alpha (0.5-50 microM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165-165 microM). The stimulation by epinephrine on progesterone production was inhibited by PGF2 alpha (5 microM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF2 alpha can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF2 alpha shows in this respect the same age dependent inhibitory pattern as in relation to LH stimulation.

Development of Sensitivity to Catecholamines in Granulosa and Luteal Cells

Catecholamines have a wide variety of physiological effects on a number of organ systems, including the nervous system, the circulation and the metabolism. The first indications that catecholamines could influence gonadal function was probably the observation that stress can depress the androgen blood level in the male (Levin et al., 1967) and the observation that the fibromuscular layer surrounding the mature follicle can increase the intrafollicular pressure and possibly promote the ovulatory process (Walles et al., 1976). The role of these effects are, however, still under debate. Catecholamines also have effects on the blood vessels of the gonads resulting in changes in blood flow (e.g. Damber et al., 1982; Selstam, 1975). The arteries are innervated (Burder, 1978), but the role of catecholamines on gonadal blood flow also remains to be investigated.

Acute stimulatory effects of luteinizing hormone-releasing hormone (LHRH) and LHRH analogues on the preovulatory rat follicle

Publisher Summary This chapter discusses acute stimulatory effects of luteinizing hormone-releasing hormone (LHRH) and LHRH analogues on the preovulatory rat follicle. In a study described in the chapter, immature Sprague–Dawley rats, 26 days old, were treated with 10 IU PMSG. This treatment resulted in the ovulation of 12–18 ova early on the morning of day 29. Ovarian follicles or granulosa cells were isolated on the morning of day 28. The follicles were incubated in Krebs–Ringer bicarbonate buffer with glucose and 1% bovine serum albumin. Granulosa cells were cultured in Eagles minimal essential medium with Hepes and 10% fetal calf serum. For the in vivo experiments, the rats were hypophysectomized on the morning of day 28. It was found that mammalian oocytes remain arrested in the prophase stage of the first meiotic division until meiosis is resumed in the preovulatory follicle following the LH surge. Oocyte meiosis can also be initiated in vitro by the addition of gonadotrophin to isolated preovulatory rat follicles.